Miho Izumikawa

Engineered Streptomyces avermitilis Host for Heterologous Expression of Biosynthetic Gene Cluster for Secondary Metabolites

An industrial microorganism, Streptomyces avermitilis, which is a producer of anthelmintic macrocyclic lactones, avermectins, has been constructed as a versatile model host for heterologous expression of genes encoding secondary metabolite biosynthesis. Twenty of the entire biosynthetic gene clusters for secondary metabolites were successively cloned and introduced into a versatile model host S. avermitilis SUKA17 or 22. Almost all S. avermitilis transformants carrying the entire gene cluster produced metabolites as a result of the expression of biosynthetic gene clusters introduced. A few transformants were unable to produce metabolites, but their production was restored by the expression of biosynthetic genes using an alternative promoter or the expression of a regulatory gene in the gene cluster that controls the expression of biosynthetic genes in the cluster using an alternative promoter. Production of metabolites in some transformants of the versatile host was higher than that of the original producers, and cryptic biosynthetic gene clusters in the original producer were also expressed in a versatile host.

Enzymatic total synthesis of enterocin polyketides

Polyketides are clinically important natural products that often require elaborate organic syntheses owing to their complex chemical structures. Here we report the multienzyme total synthesis of the Streptomyces maritimus enterocin and wailupemycin bacteriostatic agents in a single reaction vessel from simple benzoate and malonate substrates. To our knowledge, our results represent the first in vitro assembly of a complete type II polyketide synthase enzymatic pathway to natural products.

Isolation of azaspiracid-2 from a marine sponge Echinoclathria sp. as a potent cytotoxin

Azaspiracid-2 was isolated from a marine sponge Echinoclathria sp. collected off Amami-Oshima as the predominant cytotoxic constituent. A combination of HPLC using ODS, GS320, and Phenylhexyl stationary phases permitted the purification without using acid or inorganic additives in the mobile phase. Azaspiracid-2 exhibited potent cytotoxicity against P388 cells with an IC50 value of 0.72 ng/mL and caused S phase arrest on the cell cycle.

A stand-alone adenylation domain forms amide bonds in streptothricin biosynthesis

The streptothricin (ST) antibiotics, produced by Streptomyces bacteria, contain L-β-lysine ((3S)-3,6-diaminohexanoic acid) oligopeptides as pendant chains. Here we describe three unusual nonribosomal peptide synthetases (NRPSs) involved in ST biosynthesis: ORF 5 (a stand-alone adenylation (A) domain), ORF 18 (containing thiolation (T) and condensation (C) domains) and ORF 19 (a stand-alone A domain). We demonstrate that ST biosynthesis begins with adenylation of L-β-lysine by ORF 5, followed by transfer to the T domain of ORF 18. In contrast, L-β-lysine molecules adenylated by ORF 19 are used to elongate an L-β-lysine peptide chain on ORF 18, a reaction unexpectedly catalyzed by ORF 19 itself. Finally, the C domain of ORF 18 catalyzes the condensation of L-β-lysine oligopeptides covalently bound to ORF 18 with a freely diffusible intermediate to release the ST products. These results highlight an unusual activity for an A domain and unique mechanisms of crosstalk within NRPS machinery.

Sponge-derived Streptomyces producing isoprenoids via the mevalonate pathway

In the course of our screening program for isoprenoids of marine actinobacterial origin, 523 actinobacterial strains were isolated from marine samples. Actinobacteria usually use the 2-C-methyl-d-erythritol 4-phosphate pathway for the production of primary metabolites, but some have been reported to use the mevalonate (MVA) pathway for the production of isoprenoids as secondary metabolites. 3-Hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) is a key enzyme and plays an important role in the MVA pathway. Therefore, we screened strains possessing the HMGR gene from the 523 strains mentioned above and also investigated isoprenoid compounds from cultures of strains possessing HMGR genes. As a result, Streptomyces sp. SpC080624SC-11 isolated from a marine sponge, Cinachyra sp., was shown to possess the HMGR gene and produce novel isoprenoids, JBIR-46 (1), -47 (2), and -48 (3). On the basis of extensive NMR and MS analyses, the structures of 1-3 were determined to be phenazine derivatives harboring dimethylallyl moieties. Furthermore, the isoprene units of 2 and 3 were confirmed to be synthesized via the MVA pathway in a feeding experiment using [1-(13)C]acetate.

Novel in vitro protein fragment complementation assay applicable to high-throughput screening in a 1536-well format.

Protein-protein interactions (PPIs) play key roles in all cellular processes and hence are useful as potential targets for new drug development. To facilitate the screening of PPI inhibitors as anticancer drugs, the authors have developed a high-throughput screening (HTS) system using an in vitro protein fragment complementation assay (PCA) with monomeric Kusabira-Green fluorescent protein (mKG). The in vitro PCA system was established by the topological formation of a functional complex between 2 split inactive mKG fragments fused to target proteins, which fluoresces when 2 target proteins interact to allow complementation of the mKG fragments. Using this assay system, the authors screened inhibitors for TCF7/β-catenin, PAC1/PAC2, and PAC3 homodimer PPIs from 123,599 samples in their natural product library. Compound TB1 was identified as a specific inhibitor for PPI of PAC3 homodimer. TB1 strongly inhibited the PPI of PAC3 homodimer with an IC 50 value of 0.020 µM and did not inhibit PPI between TCF7/β-catenin and PAC1/PAC2 even at a concentration of 250 µM. The authors thus demonstrated that this in vitro PCA system applicable to HTS in a 1536-well format is capable of screening for PPI inhibitors from a huge natural product library. ( Journal of Biomolecular Screening 2009:970-979)

JBIR-31, a new teleocidin analog, produced by salt-requiring Streptomyces sp. NBRC 105896 isolated from a marine sponge.

JBIR-31, a new teleocidin analog, produced by salt-requiring Streptomyces sp. NBRC 105896 isolated from a marine sponge

JBIR-22, an inhibitor for protein-protein interaction of the homodimer of proteasome assembly factor 3.

Proteasome assembling chaperone (PAC) 3 acts as a homodimer and plays an important role in proteasome formation. We screened JBIR-22 (1) as an inhibitor for protein-protein interaction (PPI) of PAC3 homodimer from our natural product library using a protein fragment complementation assay (PCA) with monomeric Kusabira-Green fluorescent protein (mKG) in vitro and found that 1 exhibited potent inhibitory activity against PAC3 homodimerization. Compound 1 showed long-term cytotoxicity against the human cervical carcinoma cell line, HeLa. This is the first report of a PPI inhibitor for proteasome assembly factors.

Distribution of the 3-hydroxyl-3-methylglutaryl coenzyme A reductase gene and isoprenoid production in marine-derived Actinobacteria

During the course of our screening program to isolate isoprenoids from marine Actinobacteria, 523 actinobacterial strains were isolated from 18 marine sponges, a tunicate, and two marine sediments. These strains belonged to 21 different genera, but most were members of Streptomyces, Nocardia, Rhodococcus, and Micromonospora. Some Actinobacteria have been reported to use the mevalonate pathway for the production of isoprenoids as secondary metabolites. Therefore, we investigated whether these strains possessed the 3-hydroxyl-3-methylglutaryl coenzyme A reductase (hmgr) gene, which indicates the presence of the mevalonate pathway. As a result, six strains belonging to the genera Streptomyces (SpC080624SC-11, SpA080624GE-02, and Sp080513GE-23), Nocardia (Sp080513SC-18), and Micromonospora (Se080624GE-07 and SpC080624GE-05) were found to possess the hmgr gene, and these genes were highly similar to hmgr genes in isoprenoid biosynthetic gene clusters. Among the six strains, the two strains SpC080624SC-11 and SpA080624GE-02 produced the novel isoprenoids, JBIR-46, -47, and -48, which consisted of phenazine chromophores, and Sp080513GE-23 produced a known isoprenoid, fumaquinone. Furthermore, these compounds showed cytotoxic activity against human acute myelogenous leukemia HL-60 cells.

Pleiotropic control of secondary metabolism and morphological development by KsbC, a butyrolactone-autoregulator receptor homologue in Kitasatospora setae

ABSTRACT The γ-butyrolactone autoregulator signaling cascades have been shown to control secondary metabolism and/or morphological development among many Streptomyces species. However, the conservation and variation of the regulatory systems among actinomycetes remain to be clarified. The genome sequence of Kitasatospora setae, which also belongs to the family Streptomycetaceae containing the genus Streptomyces, has revealed the presence of three homologues of the autoregulator receptor: KsbA, which has previously been confirmed to be involved only in secondary metabolism; KsbB; and KsbC. We describe here the characterization of ksbC, whose regulatory cluster closely resembles the Streptomyces virginiae barA locus responsible for the autoregulator signaling cascade. Deletion of the gene ksbC resulted in lowered production of bafilomycin and a defect of aerial mycelium formation, together with the early and enhanced production of a novel β-carboline alkaloid named kitasetaline. A putative kitasetaline biosynthetic gene cluster was identified, and its expression in a heterologous host led to the production of kitasetaline together with JBIR-133, the production of which is also detected in the ksbC disruptant, and JBIR-134 as novel β-carboline alkaloids, indicating that these genes were biosynthetic genes for β-carboline alkaloid and thus are the first such genes to be discovered in bacteria.